1. Field of The Invention
The present invention relates to an integral multilayer element for chemical analysis. More particularly, this invention relates to an integral multilayer element for chemical analysis suitable for use in performing quantitative analysis for substances contained in aqueous liquid samples, particularly biological fluids such as blood, lymph fluid, spinal fluid, urine, etc., utilizing an oxidase enzyme reaction system which produces hydrogen peroxide upon reaction.
2. Description of Prior Arts
There have been proposed and practically used integral multilayer elements for chemical analysis (herein-after referred to often as multilayer analytical element) in the form of film or sheet as a dry analytical instrument for performing quantitative analysis on a analyte (substance to be analyzed), according to the principle that an analyte or a reaction product thereof is oxidized by an oxidas, subsequently the formed H.sub.2 O.sub.2 is involved in a dye-forming reaction in the presence of peroxidase and the formed dye is determined colormetrically.
As reagents for conducting the dye-forming reaction using oxidase and peroxidase, there are known reagents proposed by P. Trinder in the literature "Annals for Clinical Biochemistry," 6, 24-27 (1969) and their improved reagents which are generally employed in the conventional multilayer analytical element. The improved Trinder reagent is a dye-forming indicator composition containing an oxidase; peroxidase; a hydrogen donor (chromogen) such as 4-aminoantipyrine or a 4-aminoantipyrine homologue or a derivative, e.g., 4-amino-2-methyl-3-phenyl-1-(2,4,6-trichlorophenyl)-3-pyrazolin-5-one; and a coupler such as hydroxynaphthalene, e.g., 1,7-dihydroxynaphthalene, sodium 1-hydroxynaphthalene-2-sulfonate or the like. The reagents proposed by Trinder and the improved reagents (hereinafter both referred to simply as Trinder reagent) have an advantage that if the kind of the oxidase is changed, the whole remaining components can be used as the dye-forming indicator composition.
It is known that glucose oxidase, cholesterol oxidase, uricase, glycerol oxidase, etc. can be used as the oxidase. It is also known that in the dye-forming indicator composition containing the oxidase and peroxidase, dianisidine or a colorless leuco-dye such as 4,5-bis[4-(dimethylamino)phenyl]-2-(4-hydroxy-3,5-dimethoxyphenyl)imidazol e serving as a hydrogen donor can be used instead of a combination of a hydrogen donor (chromogen) and a coupler (see, U.S. Pat. No. 3,992,158, Japanese Patent Publications No. 55(1980)-25840 (U.S. Pat. No. 3,886,045 and FR 2,185,289), No. 56(1981)-45599 (U.S. Pat. No. 3,983,005), and No. 57(1982)-5519 (U.S. Pat. No. 4,089,747), etc.).
In a multilayer analytical element containing the Trinder reagent, an analyte dissolved in an aqueous liquid reaches a reagent layer where the analyte is reacted with oxygen (O.sub.2) in air under catalystic action of an oxidase to form H.sub.2 O.sub.2, and an oxidation-coupling reaction (oxidative color reaction) then proceeds under the catalytic action of peroxidase to generate or change a color. Hence, oxygen in air is an essential component for performing the reaction among a series of coupled reactions taking place in the multilayer analytical element. For this reason, there has been proposed that the oxidase is incorporated into a layer positioned above a layer containing the remaining components of the Trinder reagent (layer positioned far away from the support) to accelerate the progress of the color reaction (i.e., dye-forming reaction) and at the same time to improve the analytical accuracy (see, Japanese Patent Provisional Publication No. 57(1982)-208997 (GB 2,104,215A)). It has been found that the incorporation of the oxidase in a layer positioned above a layer containing the remaining components of the Trinder reagent has a remarkable effect of accelerating the progress of the color reaction and improving the analytical accuracy in the multilayer analytical element where a porous spreading layer and a nonporous blocking layer are provided in this order on a reagent layer containing the Trinder reagent described in Japanese Patent Provisional Publication No. 55(1980)-164356 (U.S. Pat. No. 4,292,272), etc.
However, it has been also found that in the multilayer analytical element where the oxidase is incorporated into a layer positioned above a layer containing the remaining components of the Trinder reagent, there is a disadvantage that the analytical accuracy is deteriorated by the interference of an oxidation-reduction interfering substance contained in an aqueous liquid sample spotted on a porous spreading layer. The term "oxidation reduction interfering substance" used herein refers to ascorbic acid or other material having hydrogen peroxide-decomposing activity such as catalase, hemoglobin, etc. (the term "hydrogen peroxide-decomposing activity" used herein refers to either or both of catalase activity and peroxidase activity) when the aqueous liquid sample is a biological body fluid. Further, it has been found that the above-mentioned disadvantage takes place remarkably when whole blood, hemolyzed whole blood, plasma or serum is used as the aqueous liquid sample.